SUMMARY: PROJECT 1 Corticotrophin Releasing Factor (CRF) and Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) are implicated in the stress response across species. We have shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes following psychological trauma, including posttraumatic stress disorder (PTSD), in humans. In traumatized subjects, PACAP blood levels associate with quantitative fear measures as well as with PTSD diagnosis and symptoms. We have found a sex-specific association, implicating an estrogen response element in the PAC1R gene, that predicts fear and PTSD symptoms in women. In mice, PAC1R mRNA is induced additively by fear and estrogen in both periphery and the amygdala, and recent knockout data suggest that PACAP is critical for normal CRF-dependent stress signaling. Despite these multiple lines of evidence supporting the importance of these peptide systems, the literature currently lacks clear mechanistic explanations of how they interact at a molecular level within pathways known to underlie stress, trauma, and emotional behaviors. It is now clear that the CRF and PACAP systems are interwoven and must be studied together to understand their individual and interactive roles in stress responsiveness. Project 1 is designed to operate at a deep mechanistic level to understand the role of cell-type specific CRF- and PACAP-expressing neuron populations within central amygdala (CeA), bed nucleus of the stria terminalis (BNST), prefrontal cortex (PFC), and parabrachial nucleus (PBn). Our hypothesis is that differential regulation of miRNA, mRNA, and DNA methylation, in CRF and PACAP neurons of the CeA, BNST, PFC, and PBn will identify new targets for stress-related disorders. In Aim 1, we will conduct mRNA and miRNA profiling to examine differential effects of stress. We will use CRF- and PACAP-Cre driver lines crossed with mice containing TRAP-ribosome (mRNA) and Sun-tagged markers (nuclear sorting/miRNA profiling) to examine differential effects of fear conditioning, immobilization stress, and sleep restriction stress, on miRNA/mRNA expression in these neurons within CeA, BNST, PBn, and PFC. In Aim 2, we will conduct DNA Methylation (mDNA) profiling to examine differential effects of stress. We will use the same manipulations as in Aim 1 together with nuclear sorting and whole-genome methylation arrays to examine differential effects of stress on mDNA in specific cells/regions. In Aim 3, we will perform cell-type specific manipulation of RNA expression and DNA methylation to mitigate stress. We will target a circumscribed set of genes (for Aim 3a: FKBP5; for Aim 3b: newly identified targets), using over-expression and knockdown of mRNA/miRNA, and targeted DNA (de)methylation using dCas9-TET and -Dnmt3a, within CRF and PACAP-expressing cells. This aim will explore causal relationships of these epigenetic regulatory processes within CRF/PACAP systems in mediating stress- and emotion-related behaviors. Project 1 experiments are designed based on current knowledge, but as expected with Conte Centers, we will use new discoveries from all projects to refine and optimize our objectives via the Administrative Core.